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Fig. 5 | Journal of Hematology & Oncology

Fig. 5

From: Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells

Fig. 5

DMOCPTL regulated GPX4 protein level by inducing GPX4 ubiquitination. a Western blot analyzed GPX4 expression in five TNBC cell lines. b Western blot analyzed GPX4 expression in MDA-MB-231 cells and SUM159 cells c after the treatment of DMOCPTL for 24 or 48 h at different concentrations. d MDA-MB-231 cells with DMOCPTL treatment for 48 h were collected and incubated with GPX4 antibody and corresponding FITC-conjunct second antibody. The fluorescence intensity of GPX4 expression was analyzed by flow cytometry. e The fluorescence intensity of GPX4 after the treatment of DMOCPTL for 48 h in MDA-MB-231 cells by immunofluorescence assay. f The protein level GPX4 after treatment of proteasome inhibitor MG132 at 3 h and 6 h. g MDA-MB-231 and SUM159 cell lysates were incubated with IgG or anti-Ub antibody at 4 °C for 6 h, then agarose A + G was added, co-incubated at 4 °C overnight on rotary shaker. The samples were washed with PBS and analyzed by western blot assay. h DMOCPTL was added into MDA-MB-231 cells and incubated for 24 h. The proteins were collected and incubated GPX4 antibody for 2 h on ice, then agarose G was added and co-incubated at 4 °C overnight. The ubiquitination level of GPX4 was analyzed by western blot assay with anti-Ub antibody. Analysis results represented mean ± SD, *P < 0.05, **P < 0.01

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